The goal of the proposed research is to develop a unique multi-scale mathematical model that will provide quantitative information regarding mechanisms governing nitric oxide (NO) activity in the microcirculation and to utilize innovative, real-time experiments to validate the model. The model will integrate intracellular NO production processes with extracellular, vascular and tissue transport, including the coupling of NO to O2 delivery and metabolism under normal conditions and in hypercholesterolemia. The model will help to elucidate mechanisms by which NO is produced and transported and provide greater insight into its vasodilatory role. While the importance of nitric oxide (NO) in regulating blood flow and metabolism is well established, many of the mechanisms by which NO is produced and transported have not been fully elucidated. Importantly, the various phenomena that can potentially affect the bioavailability of NO and vascular dynamics interact over a range of time and length scales. A mathematical model that transcends different spatial and time scales, coupled with in vitro and in vivo experiments, is required for understanding of system behavior. Mathematical Modeling: The development philosophy is to sequentially couple lower scale with higher scale simulation: cell-scale to vessel-scale, to vascular networks. Predictions of the simulation will be validated using our experiments and results reported in the literature. In vitr: A parallel plate laminar flow chamber, designed and constructed previously, which -- for the first time and only by our group -- enables direct, real time measurements of the kinetics of NO release under a wide range of conditions will be used for cell culture studies. The coupled effects of shear stress and altered mass transport of signaling molecules on flow- induced NO production will be investigated to isolate how they influence NO production and transport. Results will be compared with the simulations. In vivo: Experiments will be performed using the rat mesentery and apolipoprotein-E deficient mice. Local blood flow, PO2 and NO, combined with vessel diameter measurements, will be obtained from individual small arteries, arterioles, venules, and small veins under normal and abnormal physiological conditions to characterize relationships among vascular diameter, NO, blood flow, and O2 delivery. The effect of nitrite as an NO source from O2-dependent nitrite reductase activity in blood and tissue under hypoxic conditions will also be evaluated. The proposed model will lead to an improved understanding of the complete system, and set the groundwork for future research that can shed light on NO-related pathologies.